Journal: Nucleic Acids Research

Article Title: Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from Thermococcus thioreducens

doi: 10.1093/nar/gkag338

Figure Lengend Snippet: Detection of E. coli rRNA modifications by LC-MS/MS following Tth-MazF1 Cleavage. ( A ) A schematic of a Tth-MazF1 digest of a mixture of E. coli rRNA isolated from 70S ribosomes. ( B ) Fractional sequence coverage of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. ( C ) Sequence coverage maps of detected high-confidence cleavage products in Tth-MazF1 digests of E. coli rRNA. A complete list and description of the modifications are provided in . Data are a summary of two replicate experiments.

Article Snippet: Candidate genes were cloned into expression vectors as described in the section “semi-high-throughput expression and purification of maltose-binding protein (MBP)-MazF-6×His proteins.” T7 Express competent E. coli cells (NEB, C1013I) were transformed with 10 ng of each plasmid and plated on LB agar supplemented with kanamycin (50 μg/ml).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Sequencing

Journal: Cell Reports

Article Title: The insertion of an ATTTC repeat in an Alu element hyperactivates a neurodevelopmental enhancer in spinocerebellar ataxia type 37

doi: 10.1016/j.celrep.2026.117146

Figure Lengend Snippet: The DRL shows neurodevelopmental enhancer activity (A) Schematic representation of the DRL/FragT sequence in DAB1 -antisense strand. The zoom-in box (pale brown box) shows the (AAAAT) n (blue box) in the middle A-rich region of the AluJb element with left (LM) and right (RM) monomers, close to the CpG island (yellow box) and the TFBS (green box), flanked by CTCF-binding sites. The ENCODE-annotated regulatory EH38E1350977 element is represented in pink. Lines underneath represent the sequences cloned for the zebrafish transgenic lines generation, across and within the DRL sequence. (B) Stereomicroscopic images of EGFP expression in the eye and whole brain, at 24 hpf, in transgenic lines with FragT14-AS and Frag3-AS sequences cloned upstream of a promoter-less EGFP; no expression is seen in control embryos; images were processed with the Fiji software. (C) Left: representative confocal images confirming FragT14-AS and Frag3-AS expression in the eye, forebrain, midbrain, and hindbrain in these zebrafish transgenic lines at 24 hpf; maximum intensity z-projections of 14 planes; scale bars: 100 μm. Right: zoom-in images for the same zebrafish lines; orange and yellow delimitations represent eye and midbrain-hindbrain boundary close-ups, respectively; scale bars: 100 μm. (D) Left: schematic representation of the DRL sequences cloned upstream of promoter-less firefly luciferase ( luc+ ) for reporter assays in human HEK293T cells. Right: graphic representation of luciferase activity (fold change) for each sequence, as luc+/NanoLuc (Nluc) expression ratios in HEK293T cells, compared to the negative control (data are from three biological replicates, shown as the mean ± SD; one-way ANOVA test with Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001 and ∗∗∗ p < 0.001). FB, forebrain; MB, midbrain; HD, hindbrain; OV, optic vesicle; MA, mutant allele; scale bars: 100 μm. Asterisks represent autofluorescence regions. See also .

Article Snippet: As positive control, total protein extracts were extracted from HEK293T cells transfected with pCMV6-XL4-DAB1 mammalian expression vector (#SC113027, Origene) and 1 μg was loaded on SDS-PAGE gel.

Techniques: Activity Assay, Sequencing, Binding Assay, Clone Assay, Transgenic Assay, Expressing, Control, Software, Luciferase, Negative Control, Mutagenesis

Journal: Cell Reports

Article Title: The insertion of an ATTTC repeat in an Alu element hyperactivates a neurodevelopmental enhancer in spinocerebellar ataxia type 37

doi: 10.1016/j.celrep.2026.117146

Figure Lengend Snippet: DRL interaction with neural regulatory elements and silencer activity outside of the DAB1 expression domain (A) Schematics of interactions between the DRL sequence and the zebrafish midbrain-specific Z48 enhancer at 48 hpf. Top: FragT14-AS, Frag1-AS, and Frag3-AS sequences cloned upstream of a promoter-less EGFP and Z48 with schematics of their driven EGFP expression to zebrafish somites and upon Z48 interaction only to the whole midbrain. Bottom left: representative stereomicroscopic images of different EGFP expression patterns in the midbrain from FragT14-AS, Frag1-AS, and Frag3-AS interactions with the Z48 enhancer in embryos. Bottom right: graphic representation of the percentage of embryos expressing EGFP in midbrain and/or somites (shown as the mean ± SD of three replicates; EGFP-positive embryos, FragT14-AS = 238, Frag1-AS = 195, and Frag3-AS = 148, ꭕ 2 , ∗∗∗∗ p < 0.0001). (B) Left: schematic representation of the DRL sequences cloned upstream the TK promoter in the control of luciferase expression for silencer assay in HEK293T cells; right: luciferase activity in HEK293T cells, measured by the fold change in luciferase/NanoLuc (luc2/Nluc) expression ratios compared to the negative control (shown as the mean ± SD of three biological replicates; one-way ANOVA test with Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001 and ∗∗∗ p < 0.001). (C) Silencer activity of the DRL in vivo . Top: schematics of the putative silencer placed between the Z48 enhancer and the cardiac actin promoter in the reporter vector. Intermediate: graphical representation showing EGFP fluorescence intensity signal in midbrain and somites triggered by (left) FragT14-S and FragT-S-MA and (right) Frag1-S and Frag3-S sequences at 24 hpf; negative control, empty vector (data are represented as the mean ± SD; n = 60 zebrafish embryos/condition from three replicates, Kruskal-Wallis test followed by Dunn’s post hoc test; ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, and ∗ p < 0.05). Bottom: representative stereomicroscopic images of EGFP expression in zebrafish midbrain and somites driven by FragT14-S, FragT-S-MA, Frag1-S, and Frag3-S putative silencer elements; scale bars: 200 μm; arrows indicate EGFP expression in midbrain. MA, mutant allele. Asterisks show the area of autofluorescence. See also .

Article Snippet: As positive control, total protein extracts were extracted from HEK293T cells transfected with pCMV6-XL4-DAB1 mammalian expression vector (#SC113027, Origene) and 1 μg was loaded on SDS-PAGE gel.

Techniques: Activity Assay, Expressing, Sequencing, Clone Assay, Control, Luciferase, Negative Control, In Vivo, Plasmid Preparation, Fluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Togaram Ensures Axial Alignment of the Sperm Neck

doi: 10.64898/2026.04.15.718719

Figure Lengend Snippet: (A) Linear maps of human (top) and Drosophila (bottom) Togaram, Cep104, and CCDC66 depicting functional and structural domains. Domains are as indicated in the color key. (B) Both N– and C-terminally GFP-tagged Cep104 (green) binds Togaram (pink) and CCDC66 (blue). S2 cells were co-transfected with the indicated plasmids and anti-GFP co-IPs were prepared from cell lysates. Western blots of inputs and co-IPs were probed for GFP, Flag, and Myc. Arrow head colors correspond to indicated tagged protein colors. (C) Representative ExM images showing wild-type (left), cep104 null (center), and ccdc66 gRNA (right) Canoe stage spermatids with various alignment phenotypes. Spermatids were labeled for the nucleus (DAPI, blue; dotted line), the CA (Asl, magenta), and the centrioles (Ana1, green). Scale bar: ∼1 μm. (D) Quantification of wild-type (n=150), cep104 null (n=98), and ccdc66 gRNA (n=119) spermatids with various alignment phenotypes. (E) Working model in which Togaram, Cep104, and CCDC66 stabilize sperm head/neck microtubules to preserve a straight head-tail axis during the maintenance phase of spermiogenesis. Red question mark highlights a possible mechanism by which these proteins can directly regulate sperm neck stability independent of regulating microtubules.

Article Snippet: S2 cell expression vectors were generated by the Gateway cloning system (ThermoFisher Scientific) to create vectors with Actin5C promoter and the tags of interest (pAFHW, pAGW, pAMW, pAWG; F = Flag, G = GFP, M = Myc; https://emb.carnegiescience.edu/drosophila-gateway-vector-collection ). pAGW-MCS plasmid, expressing GFP alone was used as GFP control for IP experiments.

Techniques: Functional Assay, Transfection, Western Blot, Labeling